1、Designation: D5259 92 (Reapproved 2012)Standard Test Method forIsolation and Enumeration of Enterococci from Water by theMembrane Filter Procedure1This standard is issued under the fixed designation D5259; the number immediately following the designation indicates the year oforiginal adoption or, in
2、 the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a membrane filter (MF) proce-dure for the detection and enu
3、meration of the enterococcibacteria in water. The enterococci, which include Entero-coccus faecalis (E. faecalis), E. faecium, and their varieties arecommonly found in the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and notrelated to fecal pollution, enteroco
4、cci in water are an indicationof fecal pollution and the possible presence of enteric patho-gens. These bacteria are found in water and wastewater in awide range of densities. The detection limit is one colonyforming unit (CFU)/volume filtered.1.2 This test method has been used successfully withtemp
5、erate fresh and marine ambient waters, and wastewaters. Itis the users responsibility to ensure the validity of this testmethod for waters of untested types.1.3 The values stated in SI units are to be regarded as thestandard.1.4 This standard does not purport to address all of thesafety concerns, if
6、 any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. For specific hazardstatements, see Section 9.2. Referenced Documents2.1 ASTM Standards:2D
7、1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed ConduitsD3870 Practice for Establishing Performance Characteris-tics for Colony Counting Methods in Microbiology33. Terminology3.1 DefinitionsFor definitions of terms used in this te
8、stmethod, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 EnterococcusIn this test method, Enterococcus spe-cies are those bacteria that produce red to maroon colonies withblack or reddish-brown precipitate on underside, after incuba-tion on mE agar and subsequent
9、 transfer to EIA medium.Enterococci include E. faecalis, E. faecium, E. avium, and theirvariants.4. Summary of Test Method4.1 The procedure given in this test method provides adirect count of bacteria in water based on the development ofcolonies on the surface of the membrane filter.4Awater sampleis
10、 filtered through the membrane that retains the bacteria.Following filtration, the membrane containing the bacterialcells is placed on a selective, medium, mE agar, and incubatedfor 48 h at 41C, then transferred to EIA agar and held at 41Cfor 20 min. Enterococci develop as red to maroon colonies wit
11、hblack or reddish-brown precipitate on the underside of thefilter.5. Significance and Use5.1 The enterococci are indicators of the bacteriologicalquality for potable water, shellfish growing waters, ambient,and recreational waters. A direct relationship between swim-ming, associated gastroenteritis,
12、 and enterococci has beenestablished through epidemiological studies and marine andfresh water bathing beaches. These studies have led to thedevelopment of criteria that can be used to establish bathingwater standards based on established health-water qualityrelationships.1This test method is under
13、the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2012. Published August 2012. Originallyapproved in 1992. Last previous edition approved in 2006 as D5259 92 (2006).DOI: 10.1520/D5259-92R12.2F
14、or referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn. The last approved version of this historical standa
15、rd is referencedon www.astm.org.4Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W.,“Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”American Journal of Public Health , 69, 1979, pp. 690696.1Copyright ASTM International, 100 Barr Harbor
16、Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.2 Since small or large volumes of water or dilutionsthereof, can be analyzed by the membrane filter technique, awide range of levels of enterococci in water can be enumeratedand detected.6. Interferences6.1 Water with high levels
17、of colloidal or suspended mate-rials can clog the membrane filter pores and prevent filtration.Also, suspended materials cause spreading colonies that couldinterfere with target colonies and thereby prevent accuratecounting.6.2 Smaller sample size or sample dilution can be used tominimize the interf
18、erence of turbidity or high-background(non-target) bacterial densities. Replicates of smaller samplevolumes or dilutions of sample may be filtered and the resultscombined. If the membrane filter technique is not applicable,the most probable number (MPN) method for fecal strepto-cocci is recommended,
19、 with verification.6.3 In some samples, chemicals may have toxic effects onthe target organism.7. Apparatus7.1 Stereoscopic Microscope, wide-field type with magnifi-cation of 10 to 15X.7.2 Microscope Lamp, producing diffuse light from a cool,white fluorescent lamp adjusted to give maximum visibility
20、.7.3 Counting Device, hand tally or electronic.7.4 Pipet Container, stainless steel, aluminum, or borosili-cate glass, for glass pipets.7.5 Pipets, sterile tip delivery bacteriological or Mohr, glassor plastic, of appropriate volume.7.6 Graduated Cylinders, 100 to 1000 mL, covered withaluminum foil
21、or kraft paper and sterile.7.7 Membrane Filtration Units, (filter base and funnel),glass plastic or stainless steel, wrapped in aluminum foil orkraft paper and sterilized.7.8 Ultraviolet Unit, for disinfecting the filtration unit(optional).7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, foruse
22、as a vacuum source. In an emergency or in the field, a handpump or a syringe equipped with a check valve to prevent thereturn flow or air, can be used.7.10 Flask, filter, vacuum, usually 1 L, with appropriatetubing. A filter manifold to hold a number of filter bases isoptional.7.11 Forceps, straight
23、 or curved, with smooth tips to handlefilters without damage.7.12 Thermometer, checked against a National Institute ofStandards and Technology (NIST) certified thermometer, orone traceable to an NIST thermometer.7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, withtight-fitting lids.7.14 Bottles, m
24、ilk dilution, borosilicate glass, screw-capwith neoprene liners, marked at 99 mL for 1 to 100 dilutions.Dilution bottles marked at 90 mL or tubes marked at 9 mL maybe used for 1:10 dilutions.7.15 Inoculation Loops, at least 3 mm diameter, andneedles, nichrome or platinum wire, 26 B and S gage, insui
25、table holders.7.16 Incubator maintained at 41 6 0.5C.7.17 Waterbath maintained at 44 to 46C for temperingagar.7.18 Test Tubes, 150 by 20 mm, borosilicate glass or plastic.7.19 Caps, aluminum or autoclavable plastic, for 20 mmdiameter test tubes.7.20 Test Tubes, screw-cap, borosilicate glass, 125 by
26、16mm or other appropriate size.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch
27、 specifications are available.5Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.8.1.1 The agar used in preparation of culture media must beof microbiological grade. Whenever p
28、ossible, use commercialculture media as a means of quality control.8.1.2 Purity of Water Unless otherwise indicated, refer-ences to water shall be understood to mean reagent water asdefined by Type III of Specification D1193.8.1.3 Ethanol, Methanol or Isopropanol, in a small, wide-mouth container, f
29、or flame-sterilization of pipets.8.2 Membrane Filters, sterile, white, grid marked, 47 mmdiameter, with 0.45 6 0.02 m pore size or other pore sizes forwhich the manufacturer provides data demonstrating equiva-lency.8.3 Buffered Dilution Water/Buffered Rinse Water:8.3.1 Composition/Litre:Sodium dihyd
30、rogen phosphate (NaH2PO4)0.58gSodium monohydrogen phosphate (Na2HPO4)20Sodium chloride 8.50 g8.3.2 Preparation Dissolve the ingredients in 1 Lof waterin a flask and dispense in appropriate amounts for dilutions inscrew-cap bottles or culture tubes or into containers for use asrinse water, or both. A
31、utoclave after preparation at 121C (15lb pressure at sea level) for 15 min. The final pH should be 7.46 0.2.8.4 mE Agar:8.4.1 Composition of Basal Medium/Litre:Peptone 10.0 gSodium chloride 15.0 gYeast extract 30.0 gEsculin 1.0Actidione 0.05 gSodium azide 0.15 gAgar 15.0 gWater 1000 mL8.4.2 Preparat
32、ion of Basal MediumAdd 71.2 g of theabove mE basal medium to 1 L of water in a flask and heat to5“Reagent Chemicals, American Chemical Society Specifications,” Am. Chemi-cal Soc., Washington, DC. For suggestions on the testing of reagents not listed bythe American Chemical Society, see “Analar Stand
33、ards for Laboratory Chemicals,”BDH Ltd. Poole, Dorset, U.K. and the “United States Pharmacopeia.”D5259 92 (2012)2boiling until ingredients dissolve.Autoclave at 121C and 15 lbpressure for 15 min and cool in a 44 to 46C water bath.8.4.3 Reagents Added After SterilizationMix 0.25 g nali-dixic acid in
34、5 mL water, add 0.2 mL of NaOH solution (400g/L) to dissolve, and add to the litre of basal medium.Add 0.15g triphenyl tetrazolium chloride separately to the basal mediumand mix.8.4.4 Preparation of mE Agar PlatesPour the mE agarinto 50 mm petri plates to a 4 to 5 mm depth (approximately 4to 6 mL),
35、and allow to solidify. The final pH of medium shouldbe 7.1 6 0.2. Store in a refrigerator.8.5 EIA Agar:8.5.1 Composition of EIA Medium/Litre:Esculin 1.0 gFerric citrate 0.5 gAgar 15.0 gWater 1000 mL8.5.2 Preparation Add 16.5 g of dehydrated EIA mediumto 1 L of water in flask and heat to boiling unti
36、l ingredients aredissolved. Autoclave the EIA medium solution at 121C (15 lbpressure at sea level) for 15 min and cool in a 44 to 46C waterbath. After cooling, pour the medium into 50-mm petri dishesto a depth of 4 to 5 mm (approximately 4 to 6 mL and allow tosolidify. The final pH should be 7.1 6 0
37、.2 before autoclaving.Store in a refrigerator.8.6 Brain Heart Infusion (BHI) Broth:8.6.1 Composition:Calf brain infusion 200.0 gBeef heart infusion 250.0 gPeptone 10.0 gSodium chloride 5.0 gDisodium phosphate 2.5 gDextrose 2.0 gWater 1000 mL8.6.2 Preparation Dissolve 37 g of dehydrated BHI brothin 1
38、 L of water. Dispense in 8 to 10 mL volumes in screw-captubes and autoclave at 121C (15 lb pressure at sea level) for15 min. If the medium is not used the same day as prepared andsterilized, heat in boiling water bath for several min to removeabsorbed oxygen, and cool quickly without agitation, remo
39、veabsorbed oxygen, and cool quickly without agitation, just priorto inoculation. The final pH should be 7.4 6 0.2.8.7 BHI Broth with 6.5 % NaCl:8.7.1 Composition BHI broth with 6.5 % NaCl is thesame as BHI broth in 8.6 with additional NaCl.8.7.2 Preparation Dissolve 60.0 g NaCl per litre ofprepared
40、BHI broth. Since most commercially available dehy-drated media contain sodium chloride, this amount is taken intoconsideration in determining the final NaCl percentage above.8.8 BHI Agar:8.8.1 Composition BHI agar contains the same compo-nents as BHI (see 8.6) with the addition of 15.0 g of agar per
41、litre of BHI Broth.8.8.2 Preparation Add 15.0 g of agar and 37.0 g of BHIdehydrated broth to 1 L of water. Heat to boiling untilingredients are dissolved. Dispense 10 to 12 mL of medium inscrew-cap test tubes and sterilize for 15 min at 121C (15 lbpressure at sea level). Slant after sterilization. T
42、he final pHshould be 7.4 6 0.2.8.9 Bile Esculin Agar (BEA):8.9.1 Composition/Litre:Bacto beef extract 3.0 gBacto peptone 5.0 gBacto oxgall 40.0 gBacto esculin 1.0 gFerric citrate 0.5 gBacto agar 15.0Water 1000 mL8.9.2 Preparation Add 64.5 g of dehydrated BEA to 1 Lwater and heat to boiling to dissol
43、ve. Dispense in 8 to 10 mLvolumes in tubes for slants or into flasks for subsequentplating. Autoclave at 121C (15 lb pressure at sea level) for 15min. Overheating may cause darkening of the medium. Cool to44 to 46C and dispense into sterile petri plates. The final pHshould be 6.6 6 0.2. Store in a r
44、efrigerator.8.10 Gram StainPrepare according to APHA document.69. Hazards9.1 The analyst/technician must know and observe thenormal good laboratory practices and safety procedures re-quired in a microbiology laboratory while preparing, using, anddisposing of cultures, reagents, and materials, and wh
45、ileoperating sterilization and other equipment and instrumenta-tion.9.2 Mouth-pipetting is prohibited.10. Sample Collection, Preservation, and Holding Times10.1 Sampling procedures are described in detail in SectionII,Aof the USEPAmanual7and Practice D3370 and adherenceto sample preservation procedu
46、re and holding time limits iscritical to the production of valid data. Samples should not beanalyzed if these conditions are not met.10.1.1 Storage Temperature and Handling ConditionsIceor refrigerate bacteriological samples at a temperature of 1 to4C during transit to the laboratory. Use insulated
47、containers toensure proper maintenance of storage temperature. Take carethat sample bottles are not totally immersed in water duringtransit or storage.10.1.2 Holding Time LimitationsExamine samples assoon as possible after collection. Do not hold samples longerthan 6 h between collection and initiat
48、ion of analyses.11. Temperature Checks11.1 Check temperatures in incubators daily to ensureoperation within stated limits.11.2 Check thermometers at least annually against an NISTcertified thermometer or one traceable to NIST. Check mercurycolumns for breaks.6Standard Methods for Examination of Wate
49、r and Wastewater , 18th EJ.,American Public Health Association, Washington, DC, 1992, pp.9-48.7Bordner, R., Winter, J.A., and Scarpino, P. V., (eds.),“ Microbiological Methodsfor Monitoring the Environment, Water and Wastes,” EPA-600/8-78-017, U.S.Environmental Protection Agency, Office of Research and Development, Environ-mental Monitoring and Support LaboratoryCincinnati, Cincinnati, Ohio, 1978.D5259 92 (2012)311.3 See recommendations on quality control for microbio-logical analyses in the USEPAs Microbiological Methods forMonitoring the Envi
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